The analysis of the supernatant by UV–Vis spectrometry showed that each gram of P25TiO₂NP is loaded with 0.17 g of vitamin B2, after washing them. This value is coherent with the thermogram (Fig. 1), which showed a loss of 19% of weight, attributed to the thermal decomposition of vitamin B2.

Both P25TiO₂NPs (with or without vitamin B2) were not found beyond the epidermis in 99% of the analyzed TEM images (Fig. 8). This is coherent with previous findings showing that nanoparticles greater than 50 nm can not penetrate the skin, even in vivo models with movement, stretching, and friction [54]. However, in one of the zones, a few nanoparticles were observed inside a hair follicle. This could be due to the follicle exposure after the localized rupture of this physical barrier when rats were shaved in order to clean the area for cream topical administration. This finding suggests that nanoparticle-based sunscreen should not be applied on recently shaved or harmed skin, in order to avoid nanoparticle skin penetration.